HIV infections and vaccines have historically been disappointingly poor at eliciting antibodies that neutralize primary isolates, thus the identification of a highly immunogenic neutralizing epitope/s is of the highest priority. Recently several broadly crossreactive human neutralizing monoclonal antibodies have been generated, which in combination, have been shown to passively protect macaques against vaginal SIV transmission. One of these MAbs, 2G12 is unique in that it recognizes an epitope that is comprised of a cluster of up to three oligomannose chains (residues 332, 392 and 295) on the outer face of gp120. Most infected individuals do not make 2G1like neutralizing antibodies, perhaps because the high mannose glycans are trimmed as the glycoproteins traffic through the secretory pathway. To test this concept, ProcCell has used the plant expression system to produce a high-mannose form of HIV gp145 by specifically targeting synthesis of HIV gp145 to the endoplasmic reticulum using a KDEL tag which blocks exit into the Golgi and exposure to mannosidases. These high-mannose HIV Env molecules exhibited stronger reactivity with 2G12 than CHO-derived Env, suggesting important structural differences between high mannose and secreted forms of the HIV Env. The aim of the current proposal is to produce the secreted form of the gp145 (comprising a mix of complex and high mannose glycans) and to compare the biochemical, functional and immunological properties of the high-mannose form of HIV gp145 Env with both plant and cell culture-derived secreted gp120/160 molecule. Plant- derived proteins will be produced in transgenic tobacco plants and cell lines, purified and used to immunize guinea pigs to assess differences in immunogenicity of the two glycoforms. [unreadable] [unreadable] This approach represents an important new concept in optimizing vaccine efficiency by the induction of strong neutralizing 2G12-like antibodies and other potentially neutralizaing antibodies specific for yet unknown high mannose epitopes. It also highlights the usefulness of the plant expression systems in producing different glycoforms of proteins. [unreadable] [unreadable] [unreadable]